Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography. I. Analysis of bound metal ions by matrix-assisted UV laser desorption time-of-flight mass spectrometry

T. William Hutchens, Randall W. Nelson, Chee Ming Li, Tai Tung Yip

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions. Thus, LDTOF-MS is ideally suited for the rapid (3-5 min), sensitive (<1 pmol), accurate (±0.05%), and relatively high resolution (m/Δm = 300-500, full width at half maximum, where m = mass) evaluation of synthetic peptides. Further, LDTOF-MS was found to be an important tool for the characterization of peptide mixtures and peptide-metal ion interactions.

Original languageEnglish (US)
Pages (from-to)125-132
Number of pages8
JournalJournal of Chromatography A
Volume604
Issue number1
DOIs
StatePublished - Jun 26 1992

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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